Vascularization system for transplantation of endocrine organoids

Abstract

The present invention relates to a vascularization system for transplanting endocrine organoids, which is capable of maintaining the function of an isolated organoid and inducing vascularization in the vicinity of the organoid. According to the present invention, a vascularization system is formed in advance in the vicinity of an isolated organoid while toxicity to the organoid is minimized, and rapid connection to peripheral blood vessels at an implantation site is induced to facilitate the supply of materials for improving the engraftment rate and function of the organoid.

Description

Vascularization system for endocrine organoid transplantation

[0001] The present invention relates to a vascularization system for endocrine organoid transplantation capable of maintaining the function of isolated organoids and inducing vascularization around the organoids.

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0147736, filed October 31, 2023, the entire contents of which are incorporated herein by reference.

[0003] Medicines that transplant or inject cells themselves, such as stem cells, immune cells, and beta cells, are attracting attention as treatments for serious diseases. In modern medicine, commercial interest is growing in transplanting tissue replacement therapies, such as CAR-T cell therapy for blood cancer, beta cell transplantation for type 1 diabetes, and stem cell therapy, for difficult-to-treat conditions like liver cirrhosis, arthritis, and cancer.

[0004] In particular, a significant factor influencing the engraftment rate and function of organoids used as therapeutic agents is vascularization for rapid engraftment. For example, pancreatic islets have a dense network of blood vessels within the body, and this microvasculature is specialized for the supply of oxygen and nutrients, as well as glucose sensing and insulin secretion. However, during the islet isolation process, pancreatic islets lose these vascular connections. Typically, it takes about five days for new blood vessels to form around the transplant, and up to two to three weeks for functional microvasculature to fully recover. This means that transplanted pancreatic islets, with their vascular connections severed during the several weeks required for vascularization, are placed in a hypoxic state. Lack of blood flow to the transplant can damage the islets and impair their ability to regulate blood sugar.

[0005] Therefore, research is needed to improve survival rates by protecting organoids from external environmental stress, while simultaneously facilitating material exchange and angiogenesis to enhance engraftment rates and functions.

[0006] An object of the present invention is to provide a vascularization system for endocrine organoid transplantation.

[0007] To solve the above problem, the present invention provides a vascularization system for endocrine organoid transplantation including vascular endothelial cells and fibroblasts.

[0008] According to one embodiment, the vascularized system may further comprise a biocompatible hydrogel.

[0009] In one embodiment, the biocompatible hydrogel may comprise fibrinogen and thrombin. Additionally, the biocompatible hydrogel may comprise one or more of an antifibrinolytic agent and an anticoagulant.

[0010] According to one embodiment, the biocompatible hydrogel may further comprise one or more of aprotinin, tranexamic acid, nafamostat, epsilon amino caproic acid (EACA), transaminomethylcyclohexane carboxylic acid (t AMCHA), para-aminomethyl benzoic acid, a heparin derivative, a coumadin derivative, a thrombin inhibitor, and a factor Xa inhibitor.

[0011] According to one embodiment, the thickness of the vascularized system may be from 100 um to 3 mm.

[0012] According to one embodiment, the number ratio of the vascular endothelial cells and fibroblasts may be 2:1 to 10:1.

[0013] In one embodiment, the vascularized system may be for transplantation of isolated pancreatic islet organoids.

[0014] According to another embodiment of the present invention, a vascularization composition for endocrine organoid transplantation is provided, comprising a vascularization system as described above and an isolated organoid.

[0015] According to another embodiment of the present invention, there is provided a method for preparing a vascularized composition for transplantation of isolated endocrine organoids as described above,

[0016] a. A step of mixing vascular endothelial cells and fibroblasts and removing the supernatant; and

[0017] b. Provided is a method for producing a vascular composition for transplantation of isolated endocrine organoids, comprising the step of introducing an isolated organoid or an isolated spheroid into the above a. solution.

[0018] According to one embodiment, the present invention may further comprise a step comprising: c. adding fibrinogen and an antifibrinolytic agent; and d. rapidly mixing after adding thrombin.

[0019] According to another embodiment of the present invention, there is provided an isolated endocrine organoid vascularized with a vascularization system for endocrine organoid transplantation as described above.

[0020] Specific details of other embodiments of the present invention are included in the detailed description below.

[0021] According to the present invention, a vascularization system is formed in advance around an isolated organoid while minimizing toxicity to the organoid, and rapid connection with peripheral blood vessels at the transplantation site is induced, thereby facilitating the supply of materials to improve the engraftment rate and function of the organoid.

[0022] Figure 1 is a schematic diagram illustrating an overview of a vascularization system for organoid transplantation.

[0023] Figure 2 is a photograph observing the porous structure of the vascular system.

[0024] Figure 3 is a photograph showing a vascularized system loaded with cell clusters.

[0025] Figure 4 shows the results of analyzing glucose-dependent insulin secretion ability according to the presence or absence of vascularization.

[0026] Figure 5 shows the results of long-term blood sugar analysis of the vascular system.

[0027] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0028] Hereinafter, a vascularization system for endocrine organoid transplantation, an isolated endocrine organoid transplantation vascularization composition, and a method for manufacturing the same according to an embodiment of the present invention will be described in more detail.

[0029] The present invention introduces a vascularization system onto the surface of an organoid, enabling the pre-existing formation of a vascular structure around the organoid prior to transplantation. Consequently, after transplantation, the vascular structure surrounding the organoid rapidly connects with the blood vessels at the transplant site, thereby improving engraftment rates and function.

[0030] The present invention provides a vascularization system for endocrine organoid transplantation comprising vascular endothelial cells and fibroblasts.

[0031] According to one embodiment, the vascularization system of the present invention may further comprise a biocompatible hydrogel. The biocompatible hydrogel may include a collagen hydrogel, an extracellular matrix-based hydrogel including Matrigel, and the like.

[0032] According to one embodiment, the hydrogel may comprise fibrinogen and thrombin. In addition, the hydrogel may comprise one or more of an antifibrinolytic agent and an anticoagulant. Specifically, for example, the hydrogel may further comprise one or more of aprotinin, tranexamic acid, nafamostat, epsilon amino caproic acid (EACA), trans-aminomethylcyclohexane carboxylic acid (t AMCHA), para-aminomethyl benzoic acid, a heparin derivative, a coumadin derivative, a thrombin inhibitor, and a factor Xa inhibitor.

[0033] In addition to the types described above, additional factors can be added to preform vascular structures in vitro, or functionality can be enhanced through genetic modification. Specifically, additional factors can include fibrin glue, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor β (TGTβ), and VEGF mRNA. Carriers such as lipid nanoparticles (LNPs) can be utilized for the delivery of these factors.

[0034] The hydrogel of the present invention may be pre-vascularized prior to transplantation by including the components described above.

[0035] In one aspect, the present invention provides a vascularization system for endocrine organoid transplantation and a vascularization composition for organoid transplantation, comprising isolated organoids as described above. The isolated organoids may include single cells, cell clusters, artificial organs, and the like.

[0036] According to another aspect of the present invention, a vascularized organoid is provided using the vascularization composition for organoid transplantation as described above. The vascularized organoid may be one in which a single cell or two or more cells are vascularized, or may be a cell cluster or a surface of an artificial organ that is vascularized, or may be isolated. For example, the vascularizable organoid may be an endocrine organoid, or an organoid derived from stem cells, and specifically, may include one or more of β-cells, pancreatic islets, adrenal glands, muscle cells, fibroblasts, and blood cells.

[0037] According to another aspect of the present invention, a vascularization composition for organoid transplantation is provided, comprising a vascularization system for endocrine organoid transplantation as described above and an isolated organoid.

[0038] According to another embodiment of the present invention, a method for preparing a vascular composition for transplantation of isolated endocrine organoids is provided, comprising the steps of: a. mixing vascular endothelial cells and fibroblasts and removing the supernatant; and b. adding isolated organoids or isolated spheroids to the solution a.

[0039] According to one embodiment, c. adding fibrinogen and an antifibrinolytic agent; and

[0040] d. A step may further include a step of rapidly mixing after adding thrombin.

[0041] According to one embodiment, the vascularized system can comprise a content of endothelial cells and fibroblasts in a number ratio of from 2:1 to 10:1, for example from 3:1 to 8:1, from 3:1 to 6:1, or from 4:1 to 5:1.

[0042] In one embodiment, the vascularization system may include endothelial cells, fibroblasts, fibrinogen (or fibrin), thrombin, and aprotinin. For example, aprotinin may be included at 3 to 8 TIU / ml (45 μl). In one embodiment, the fibroblasts may be removed prior to transplantation.

[0043] According to one embodiment, the vascularization system may further comprise one or more of an antifibrinolytic agent and an anticoagulant. Specifically, for example, the vascularization system may further comprise one or more of aprotinin, epsilon amino caproic acid (EACA), trans-aminomethylcyclohexane carboxylic acid (t AMCHA), para-aminomethyl benzoic acid, a heparin derivative, a coumadin derivative, a thrombin inhibitor, and a factor Xa inhibitor.

[0044] According to a specific embodiment, the method may include mixing vascular endothelial cells and fibroblasts in a number ratio of 2:1 to 10:1 to form a vascularization system for endocrine organoid transplantation, washing with a large amount of PBS, and centrifuging. After removing all the supernatant, the method may include adding washed organoids or spheroids, and adding 2X fibrinogen and aprotinin to the solution after removing the supernatant. Finally, the method may include adding thrombin, carefully and quickly mixing the organoids or spheroids so as not to break them, and then placing them in a mold. After solidifying at room temperature for about 10 to 30 minutes, placing them in an incubator for an additional 10 to 30 minutes, and then adding a medium and culturing them for about 5 days. At this time, the components used are 1 X 10 vascular endothelial cells based on a total volume of 500 ul. 6 Dog, fibroblasts 2 X 10 5 It may contain 450ul of 2% fibrinogen (+ PBS) (final concentration 1%), 45ul of aprotinin at 3 to 8 TIU / ml, and 4.5ul of thrombin at 200 U / ml.

[0045] According to one embodiment, the thickness of the vascularization system for endocrine organoid transplantation according to the present invention can be formed to be 100 um to 3 mm, for example, 100 um to 2 mm, or 100 um to 1 mm, and the thickness can be appropriately adjusted depending on the intended use, application environment, etc.

[0046] According to one embodiment, the vascularization composition for endocrine organoid transplantation can be imaged by introducing a labeling substance such as a fluorescent molecule or a luminescent molecule. In addition, drugs, growth factors, etc. can be introduced and locally delivered to the target of application. For example, imaging can be achieved by introducing a labeling substance such as a chromogenic enzyme such as peroxidase, alkaline phosphatase, a radioisotope, a colloid, PE (phycoerythrin), FCA (fluorescein carboxylic acid), HRP, TAMRA, FITC (poly L-lysine-fluorescein isothiocyanate), RITC (rhodamine-B isocyanate), rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, quantum dots, etc. Additionally, for example, antibody epitopes, substrates, cofactors, inhibitors, or affinity ligands can be introduced.

[0047]

[0048] As described above, the vascularization system for endocrine organoid transplantation of the present invention can rapidly establish a vascular structure around the organoid in vitro, thereby facilitating rapid connection with the blood vessels at the transplant site upon in vivo transplantation. Consequently, material supply to the organoid via blood vessels is rapidly achieved, thereby improving engraftment rates and function.

[0049]

[0050] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0051] Example 1: Formation of a vascularized system for endocrine organoid transplantation.

[0052] Based on the total volume of 500ul, 1 X 10 vascular endothelial cells 6 Dog, fibroblasts 2 X 10 5 The vascularization system was introduced using 450ul of 2% fibrinogen (in PBS) (final concentration 1%), 45ul of aprotinin (3 TIU / ml), and 4.5ul of thrombin (200 U / ml).

[0053] First, vascular endothelial cells and fibroblasts were mixed at a content ratio of 5:1 vascular endothelial cells:fibroblasts, washed with a large amount of PBS, and then centrifuged.

[0054] After removing all the supernatant, washed β-cell organoids were added. 2X fibrinogen was added to the solution, followed by aprotinin. Finally, thrombin was added, and the spheroids were carefully and rapidly mixed so as not to break up, and then placed in a mold. After solidifying at room temperature for about 10 minutes, they were placed in a 37°C incubator for an additional 10 minutes, medium was added, and cultured for about 5 days. An overview of the vascularization system for organoid transplantation of the present invention is illustrated in Fig. 1. In the schematic diagram of the preparation of the organoid vascularization system (PV-gel) in which the vascularization system illustrated in Fig. 1 is introduced, all materials were filtered before use, and the agarose mold was removed one hour after gelation was completed. The medium was changed every 12 hours, and each gel structure consisted of 1,200 spheroids (1,000 cells per spheroid).

[0055]

[0056] Experimental Example 1: Porous Structural Analysis of a Vascularization System for Endocrine Organoid Transplantation

[0057] Hydrogel structures were formed by mixing fibrinogen and aprotinin in a ratio of 1:1, 2:1, or 4:1, respectively. 2X fibrinogen was completely dissolved in the isotonic solution, and then thrombin-containing serum was mixed with different ratios of aprotinin. The gel structures were placed at room temperature for 15 minutes, and then incubated at 37°C for 30 minutes to complete gelation.

[0058] Scanning electron microscope (SEM) images of the porous structure of vascularized hydrogels according to the composition ratio of fibrinogen and aprotinin are shown in Figure 2.

[0059]

[0060] Experimental Example 2: Analysis of a vascularized system loaded with cell clusters

[0061] Immunostaining images of the vascularized system structures were observed 5 days after gelation of the vascularized system for endocrine organoid transplantation according to Example 1. The content ratio of fibrinogen and aprotinin in the vascularized system used for image observation was 4:1.

[0062] Optical microscopy images and immunofluorescence staining images of a vascularization system equipped with cell clusters are shown in Fig. 3. The upper left of Fig. 3 is a phase contrast image of a spheroid vascularized PV-gel (Sph / PV-gel) with a vascularization system introduced, and the lower left of Fig. 3 is a phase contrast image of a non-vascularized spheroid gel (Sph-gel). The right side of Fig. 3 is a confocal laser microscope image of the PV-gel. Endothelial cells (EC) are indicated in green, insulin (β-cells) in red, and nuclei in blue (scale bar: 100 um).

[0063]

[0064] Experimental Example 3: Analysis of Glucose-Dependent Insulin Secretion in a Vascularized System for Endocrine Organoid Transplantation

[0065] The glucose-dependent insulin secretion ability of the pancreatic islet clusters loaded into the vascularization system for endocrine organoid transplantation according to Example 1 was confirmed according to the presence or absence of vascularization. The content ratio of fibrinogen and aprotinin in the vascularization system used was 4:1.

[0066] Hydrogels containing vascularized pancreatic islet clusters were implanted subcutaneously in C57BL / 6J mice.

[0067] The analysis results are shown in Fig. 4. Fig. 4, A shows the results of confirming the insulin levels of spheroid gel (Sph-gel) and spheroid vascularized PV-gel, and Fig. 4, B shows the results of comparing the insulin levels in a high-glycemic solution with the insulin levels in a low-glycemic solution. Error bars represent the mean ± standard deviation (SD).

[0068]

[0069] Experimental Example 4: Long-term Blood Glucose Analysis in the Vascular System

[0070] Using the same vascularization system as in Experimental Example 3, islet clusters with vascularization systems were transplanted subcutaneously into type 1 diabetic mice, and their glycemic control effects were confirmed. Long-term non-fasting blood glucose analysis results of the transplanted islet clusters are shown in Figure 5. It was confirmed that PV-gel with vascularization systems maintained long-term lower fasting blood glucose levels compared to non-vascularized Sph-gel.

[0071]

[0072] As confirmed by the above results, introducing a vascularization system onto the organoid surface allows for the pre-formation of a microvascular structure around the transplanted organoid, and rapid connection with the blood vessels at the transplant site after transplantation. Consequently, material is rapidly supplied to the transplanted organoid via blood vessels, significantly improving engraftment rates and functionality.

[0073] In addition, while conventional vascularization techniques require vascularization devices for transplantation costing tens of millions to hundreds of millions of won to perform, the transplantation process using the vascularization system for endocrine organoid transplantation of the present invention can be performed at the laboratory level or in an operating room, thereby reducing costs and enabling the introduction of the technology without requiring significant expertise.

[0074] The above description is merely an illustrative illustration of the technical idea of ​​the present invention, and those skilled in the art can make various modifications and variations without departing from the essential characteristics of the present invention. In addition, the embodiments disclosed in the present invention are not intended to limit the technical idea of ​​the present invention, but rather to explain it, and the scope of the technical idea of ​​the present invention is not limited by these embodiments. The protection scope of the present invention should be interpreted by the following claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

Claims

1. Vascularization system for endocrine organoid transplantation containing vascular endothelial cells and fibroblasts.

2. In paragraph 1, A vascularization system for endocrine organoid transplantation, wherein the vascularization system further comprises a biocompatible hydrogel.

3. In paragraph 2, A vascularization system for endocrine organoid transplantation, wherein the biocompatible hydrogel comprises fibrinogen and thrombin.

4. A vascularization system for endocrine organoid transplantation, wherein the biocompatible hydrogel comprises at least one of an antifibrinolytic agent and an anticoagulant in the second paragraph.

5. In paragraph 2, A vascularization system for endocrine organoid transplantation, wherein the biocompatible hydrogel further comprises at least one of aprotinin, tranexamic acid, nafamostat, epsilon amino caproic acid (EACA), transaminomethylcyclohexane carboxylic acid (t AMCHA), para-aminomethyl benzoic acid, a heparin derivative, a coumadin derivative, a thrombin inhibitor, and a factor Xa inhibitor.

6. In paragraph 1, A vascularization system for endocrine organoid transplantation, wherein the thickness of the vascularization system is 100 um to 3 mm.

7. In paragraph 1, A vascularization system for endocrine organoid transplantation, wherein the number ratio of the vascular endothelial cells and fibroblasts is 2:1 to 10:

1.

8. In paragraph 1, A vascularization system for endocrine organoid transplantation, wherein the vascularization system is for transplantation of isolated pancreatic islet organoids.

9. A vascularization composition for endocrine organoid transplantation, comprising a vascularization system according to paragraph 1 and an isolated organoid.

10. A method for producing a vascular composition for transplantation of isolated endocrine organoids according to Article 9, a. A step of mixing vascular endothelial cells and fibroblasts and removing the supernatant; and b. A method for producing a vascularized composition for transplantation of isolated endocrine organoids, comprising the step of introducing an isolated organoid or an isolated spheroid into the above a. solution.

11. In paragraph 10, c. a step of adding fibrinogen and an antifibrinolytic agent; and d. A method for producing a vascular composition for transplantation of isolated endocrine organoids, further comprising a step of rapidly mixing after adding thrombin.

12. An isolated endocrine organoid vascularized with a vascularization system for endocrine organoid transplantation according to paragraph 1.

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